Human Papillomavirus, Tobacco, and Poor Oral Hygiene Can Act Synergistically, Modulate the Expression of the Nuclear Factor Kappa B Signaling Pathway for the Development and Progression of Head and Neck Cancer in the Pakistani Population
Head and neck cancers (HNCs) represent a heterogeneous group of malignancies that arise in various anatomical sites within the upper aerodigestive tract, including the oral cavity, oropharynx, hypopharynx, nasopharynx, and larynx. These cancers are a significant global health burden, accounting for over 650,000 cases and 330,000 deaths annually. In South Asian countries, particularly in Pakistan, oral cancer is the most prevalent form of HNC, with more than 90% of cases originating from squamous cells of the oral cavity, oropharynx, and larynx. The development and progression of HNC are influenced by a combination of environmental and genetic risk factors, including tobacco use, alcohol consumption, ultraviolet radiation exposure, and viral infections, particularly human papillomavirus (HPV).
The nuclear factor kappa B (NF-kB) signaling pathway plays a critical role in regulating cell signaling pathways involved in inflammation, apoptosis, cell proliferation, and angiogenesis. NF-kB is typically maintained in an inactive state in the cytoplasm by inhibitory proteins such as IkBa. Upon activation by various stimuli, including cytokines, bacterial or viral infections, and stress signals, NF-kB translocates to the nucleus and induces the expression of numerous downstream genes. Dysregulation of the NF-kB pathway is frequently observed in various malignancies, including lymphoma, leukemia, myeloma, and cancers of the colon, pancreas, breast, and cervix. In the context of HNC, HPV infection has been identified as a significant etiological factor, particularly in oropharyngeal carcinoma. High-risk HPV genotypes, such as HPV16 and HPV18, produce early proteins E6 and E7, which deregulate cell cycle control and activate NF-kB signaling, contributing to carcinogenesis.
The present study aimed to investigate the prevalence of high-risk HPV genotypes and the expression of NF-kB signaling pathway proteins in HNC patients in the Pakistani population. The study also explored the synergistic effects of tobacco use and poor oral hygiene on HPV infection and NF-kB pathway modulation in HNC development and progression.
The study was conducted on 152 HNC tumor samples collected from various hospitals in Pakistan. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue samples using a commercial DNA extraction kit. HPV detection and genotyping were performed using polymerase chain reaction (PCR) with general and type-specific primers, followed by agarose gel electrophoresis. Immunohistochemistry (IHC) was employed to detect the expression of HPV E6 protein and NF-kB pathway proteins, including p50, p65, and IkBa inhibitor, in tumor tissues.
PCR analysis revealed that 24.3% (37/152) of HNC cases were positive for HPV infection. Among the HPV-positive cases, 86.5% (32/37) were tobacco users, while 66.9% (77/115) of HPV-negative cases were tobacco users. A significant association between HPV positivity and tobacco use was observed, with an odds ratio (OR) of 0.310 and a 95% confidence interval (CI) of 0.110 to 0.870. Furthermore, 78.3% (29/37) of HPV-positive patients had poor oral hygiene, compared to 21.6% (8/37) with good oral hygiene. The logistic regression analysis indicated that age, tobacco use, and oral hygiene were significant predictors of HPV-associated HNC.
HPV genotyping results showed that HPV16 was the predominant genotype, accounting for 64.8% (24/37) of HPV-positive cases, followed by HPV18 at 35.2% (13/37). IHC staining confirmed the presence of HPV E6 protein in tumor tissues, with strong nuclear and cytoplasmic staining observed in HPV-positive cases. Expression analysis of NF-kB pathway proteins revealed that p50 and p65 were predominantly localized in the nucleus, while IkBa inhibitor was found in the cytoplasm. The expression levels of p50 and p65 were significantly higher in HPV-positive cases compared to HPV-negative cases, with strong staining intensity observed in 81% (30/37) and 67% (25/37) of HPV-positive cases, respectively. In contrast, IkBa inhibitor showed low expression in HNC cases, with weak immunostaining observed in 46% (70/152) of samples.
The study also examined the correlation between NF-kB pathway protein expression and various clinicopathological characteristics of HNC patients. A positive correlation was observed between p50 and p65 expression, while a negative correlation was found between p50 and IkBa inhibitor expression. Additionally, a significant positive correlation was observed between tobacco use and poor oral hygiene, as well as between primary tumor stage and tumor grade.
The findings of this study highlight the synergistic role of HPV infection, tobacco use, and poor oral hygiene in modulating the NF-kB signaling pathway and contributing to the development and progression of HNC in the Pakistani population. The high prevalence of HPV16 in HNC cases underscores the importance of HPV vaccination and early detection strategies in reducing the burden of HPV-associated HNC. Furthermore, the study emphasizes the need for public health interventions to address tobacco use and improve oral hygiene practices as part of comprehensive cancer prevention and control programs.
In conclusion, this study provides valuable insights into the molecular mechanisms underlying HPV-associated HNC and the role of the NF-kB signaling pathway in carcinogenesis. The findings underscore the importance of integrating molecular biomarkers into clinical practice for the early detection and management of HNC. Additionally, the study highlights the need for further research with larger sample sizes to elucidate the complex interplay between HPV infection, environmental risk factors, and genetic pathways in HNC development and progression.
doi.org/10.1097/CM9.0000000000001970
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